Synthesis by bacteria has long been the only means for production of many of the more complex biopolymers. Only recently have pathways for the synthesis of these polymers been determined. Much effort has gone into the isolation of the various enzymes and cofactors involved in these pathways. Regulation of their expression has largely been empirical, i.e., the concentration of nutrients or other factors such as oxygen level have been altered and the effect on polymer production and composition measured.
In order to have control over the production of these complex biopolymers, and to modify them in a specific fashion, it is necessary to design a system for determining the chemical steps required for their synthesis; to isolate and characterize the proteins responsible for these chemical steps; to isolate, sequence, and clone the genes encoding these proteins; and to identify, characterize, and utilize the mechanisms for regulation of the rate and level of the expression of these genes.
Polyhydroxybutyrate, a commercially useful complex biopolymer, is an intracellular reserve material produced by a large number of bacteria. Poly-beta-hydroxybutyrate (PHB), the polymeric ester of D(xe2x88x92)-3-hydroxybutyrate, was first discovered in Bacillus megaterium in 1925. Both the chemical and physical properties of this unique polyester have made it an attractive biomaterial for extensive study. PHB has a variety of potential applications, including utility as a biodegradable/thermoplastic material, as a source of chiral centers for the organic synthesis of certain antibiotics, and as a matrix for drug delivery and bone replacement. In vivo, the polymer is degraded internally to hydroxybutyrate, a normal constituent of human blood.
The enzymatic synthesis of the hydrophobic crystalline PHB granules from the C2 biosynthon acetyl-CoA has been studied in a number of bacteria. Three enzymes: beta ketothiolase, acetoacetyl-CoA reductase and PHB polymerase, are involved in the conversion of acetyl-CoA to PHB.
Thiolases are ubiquitous enzymes which catalyze the synthesis and cleavage of carbon-carbon bonds and thus occupy a central role in cellular metabolism. Different thiolase enzymes are involved in terpenoid, steroid, macrolide and other biosynthetic pathways as well as the degradation of fatty acids. In Z. ramigera, the condensation of two acetyl-CoA groups to form acetoacetyl-CoA is catalyzed by beta-ketothiolase. The acetoacetyl-CoA is then reduced by an NADP-specific reductase to form D(xe2x88x92)-beta-hydroxybutyryl-CoA, the substrate for PHB polymerase.
Beta-Ketothiolase (acetyl-CoA-CoA-C-acetyl-transferase, E.C. 2.3.1.9) has been studied in A. beijerinckii (Senior and Dawes, Biochem. J., 134, 225-238 (1973)), A. eutrophus (Oeding and Schlegel, Biochem. J., 134, 239-248 (1973)), Clostridium pasteurianum (Bernt and Schlegel, Arch. Microbiol., 103, 21-30 (1975)), and Z. ramigera (Nishimura et al., Arch. Microbiol., 116, 21-27 (1978)). The cloning and expression of the Z. ramigera acetoacetyl-CoA reductase genes was described in U.S. Ser. No. 067,695. This gene was then used as a hybridization probe to isolate the reductase gene from other bacterial species, including Alcaligenes eutrophus and Nocardia.
The reductase involved in PHB biosynthesis in Z. ramigera is stereospecific for the D(xe2x88x92)-isomer of hydroxybutyryl-CoA and uses NADP(H) exclusively as a cofactor. The best characterized Acetoacetyl-CoA reductase is that from Zoogloea, described by Saito et al., Arch. Microbiol., 114, 211-217 (1977) and Tomita et al., Biochemistry of Metabolic Processes, 353, D. Lennon et al., editors (Elsevier, Holland, 1983). This NADP-specific 92,000 molecular weight enzyme has been purified by Fukui, et al., Biochim. Biophys. Acta 917, 365-371 (1987) to homogeneity, although only in small quantities. As described in U.S. Ser. No. 067,695, the beta-ketothiolase enzyme from Z. ramigera has now been cloned, expressed and the product thereof purified to homogeneity. The cloned gene was used to identify and isolate the corresponding beta-ketothiolase gene in other bacterial species, including Alcaligenes eutrophus and Nocardia.
The PHB polymerase in Z. ramigera is stereospecific for D-beta-hydroxybutyryl CoA. Synthetases from other bacteria such as A. eutrophus can utilize other substrates, for example, D-beta-hydroxyvaleryl CoA, since addition of propionate into A. eutrophus cultures leads to incorporation of C5 and C4 units into a PHB/HV copolymer. Griebel and Merrick, J. Bacteriol., 108, 782-789 (1971) separated the PHB polymerase from native PHB granules of B. megaterium, losing all of the enzyme activity in the process. They were able to reconstitute activity only by adding PHB granules to one of two fractions of the protein. More recently, Fukui et al., Arch. Microbiol., 110, 149-156 (1976) and Tomita et al. (1983), investigated this enzyme in Z. ramigera and partially purified the non-granule bound PHB polymerase. A method for cloning, expressing and using the product thereof in the synthesis of novel polymers was described in U.S. Ser. No. 067,695.
A whole range of polyhydroxalkanoate (PHA) storage polymers has been found to be produced by bacteria, including A. eutrophus and P. oleovarans. The PHA polymers are heteropolymers of the D-isomer of xcex2-hydroxyalkanoates with the variation occurring in the length of, the side chains (CH3-CH8H17). For example, when grown in the presence of 5-chloropentanoic acid, A. eutrophus incorporates 3-hydroxybutyrate, 3-hydroxyvalerate and 5-hydroxyvalerate into the polymer.
Given the extremely high yields of this polymer obtainable through classic fermentation techniques, and the fact that PHB and PHA of molecular weight greater than 10,000 is useful for multiple applications, it is desirable to develop new PHB-like biopolymers to improve or create new applications.
The production of poly-beta-hydroxyalkanoates, other than PHB, by monocultures of A. eutrophus and Pseudomonas oleovorans was reported by deSmet, et al., in J. Bacteriol., 154, 870-878 (1983). In both bacteria, the polymers were produced by controlled fermentation. A. eutrophus, when grown on glucose and propionate, produces a heteropolymer of PHB-PHV, the PHV content reaching approximately 30%. P. oleovorans produces a homopolymer of poly-beta-hydroxyoctanoate when grown on octane. Nocardia has been reported to form copolymers of PHB-PH-2-butenoate when grown on n-butane. Determination of the final composition of 3-hydroxybutyrate polymers by controlled fermentation using selected substrates is also disclosed in U.S. Pat. No. 4,477,654 to Holmes et al.
With the availability of a variety of enzymes varying as to their substrate specificity and techniques for expressing the genes encoding the enzymes in other hosts, especially plants, it is possible to provide an economic, biodegradable alternative to the presently available plastics derived from petroleum, especially polypropylene.
It is therefore an object of the present invention to provide further enzymes for use in a method for synthesis of complex biopolymers, particularly PHB, PHA and similar polymers.
It is a further object of this invention to isolate, sequence, and clone additional genes encoding these proteins for polymer synthesis, as well as means for regulation of the rate and level of the expression of these genes.
It is another object of the present invention to provide purified proteins expressed from the genes encoding the proteins for synthesis of polyhydroxybutyrate and polyhydroxyalkanoate.
It is a further object of the present invention to provide methods for using these proteins and regulatory sequences to create novel biopolymers having polyester backbones.
It is a still further object of the present invention to provide an economic source of biodegradable polyhydroxyalkanoates and novel related polymers, using both bacterial and plant cells for production.
A method for controlling and modifying biopolymer synthesis by manipulation of the genetics and enzymology of synthesis of polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA) polyesters at the molecular level in procaryotic and eukaryotic cells, especially plants.
Examples demonstrate the isolation, characterization, and expression of the genes involved in the production of PHB and PHA polymers. Genes encoding the enzymes in the PHB and PHA synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB polymerase or PHA polymerase) from Zoogloea ramigera strain I-16-M, Alcaligenes eutrophus, Nocardia salmonicolur, and Psuedomonas olevarans were identified or isolated and expressed in a non-PHB producing organism, E. coli. 
In a preferred embodiment using bacterial cells, the polymer is made in A. eutrophus due its capacity for accumulating PHB up to 70 to 80% dry cell weight under conditions of nitrogen or phosphate limitation. In another embodiment, the genes are introduced into plant cells for expression and synthesis of PHB, PHA, and novel polymers. Specific modifications to the polymers include variation in the chain length of the polymers and incorporation of different monomers into the polymers to produce co-polymers with different physical properties.